COMMUNICATION

Aldehyde and ketone synthesis via P450-catalyzed oxidative

deamination of alkyl azides

Simone Giovani, Hanan Alwaseem and Rudi Fasan*[a]

Abstract: Heme-containing proteins have recently attracted

increasing attention for their ability to promote synthetically valuable

transformations not found in nature. Following the recent discovery

that engineered variants of myoglobin can catalyze the direct

conversion of organic azides to aldehydes, we investigated the azide

oxidative deamination reactivity of a variety of hemoproteins featuring

different heme coordination environments. Our studies show that

although several heme-containing enzymes possess basal activity in

this reaction, an engineered variant of the bacterial cytochrome P450

CYP102A1 constitutes a particularly efficient biocatalyst for promoting

this transformation, exhibiting a broad substrate scope along with high

catalytic activity (up to 11,300 TON), excellent chemoselectivity, and

enhanced reactivity toward secondary alkyl azides to yield ketones.

Mechanistic studies and Michaelis-Menten analyses provided insights

into the mechanism of the reaction and the impact of active site

mutations on the catalytic properties of the P450. Altogether, these

studies demonstrate that engineered P450 variants represent

promising biocatalysts for the synthesis of aryl aldehydes and ketones

via the oxidative deamination of alkyl azides under mild reaction

conditions.

1. Introduction

Cytochrome P450s constitutes a superfamily of iron-dependent,

heme-containing oxygenases which play an important role in drug

metabolism, biodegradation, and biosynthesis of secondary

metabolites.[1] These enzymes have received significant attention for

their ability to hydroxylate aliphatic and aromatic C−H bonds, a

challenging reaction to achieve by chemical means[2] Furthermore,

P450s are known to promote a variety of other oxidative

transformations including heteroatom dealkylation, carbon-carbon

bond cleavage, rearrangement reactions, and Baeyer-Villiger

reactions.[3] More recently, the scope of cytochrome P450s has been

extended to a number of important, 'non-native' reactions useful for

the construction of carbon−carbon,[4] carbon−nitrogen,[5] and nitrogen-

sulfur[6] bonds.

Our group has recently reported that the heme-containing protein

myoglobin (Mb) constitutes a promising scaffold for promoting a

variety of synthetically useful transformations mediated by metal-

carbenoid and -nitrenoid species.[7] In particular, previous work

showed that engineered variants of this hemoprotein can catalyze the

oxidative deamination of organic azides to generate aldehydes.[8] In

comparison to classical methods involving alcohol oxidation with toxic

chromium-based reagents, this transformation provides a convenient

approach to the synthesis of aldehydes starting from a non-

oxygenated functional group and using readily accessible alkyl azides.

Furthermore, synthetic catalysts (e.g., MoO2(S2CNEt2)2) currently

available to promote this reaction suffer from poor catalytic efficiency

(20-200 turnovers) and require harsh reaction conditions (reflux in

toluene/water mixture).[9] In the interest of comparing and contrasting

the reactivity of hemoproteins featuring different heme coordination

environments in the context of non-native reactions, we investigated

and report here the azide oxidative deamination reactivity of a panel

of different heme-containing enzymes, including a catalase, a

peroxidase, and wild type and engineered variants of a bacterial

cytochrome P450. These studies led to the identification of an

engineered variant of CYP102A1 as a superior biocatalyst for the

conversion of a broad range of aryl-substituted alkyl azides to the

corresponding aldehydes and ketones. In addition, insights into the

mechanism and catalytic properties of this enzyme were gained via a

combination of kinetic studies and isotopic labeling experiments.

To investigate the scope of hemoprotein-catalyzed azide-to-

aldehyde oxidation, we initially selected a panel of different heme-

containing enzymes consisting of bovine catalase (Cat), horseradish

peroxidase (HRP), and the bacterial cytochrome CYP102A1[10] (also

known as P450BM3)[11]. These enzymes feature a significantly different

heme coordination environment compared to each other and to the

previously investigated myoglobin (Mb). In Cat and CYP102A1, the

amino acid ligand involved in coordinating the heme iron is a tyrosine

and cysteine residue, respectively[12], as opposed to histidine in Mb[13].

The heme group in HRP is also bound through a histidine residue,[14]

but a strong H-bonding interaction between this proximal His and a

neighboring aspartic acid residue confers a considerable anionic

character to the former, a structural feature that is not present in Mb.

Finally, hemoglobin (Hb) was also considered to evaluate the effect of

tetrameric structure on the target reactivity as opposed to the

monomeric structure of Mb.

As a model reaction, the conversion of benzyl azide 1 to

benzaldehyde 2a under anaerobic conditions in phosphate buffer (pH

7.0) and in presence of sodium dithionite (Na2S2O4) as a reductant

(Figure 1a) was used to assess the relative efficiency of these

hemoproteins in promoting azide oxidative deamination. Under these

conditions, Cat, HRP and Hb show only low to moderate reactivity,

supporting about 5, 63 and 195 turnovers (TON), respectively (Figure

1b-c). These TON values are lower or comparable to those achieved

with free hemin under similar reaction conditions (~100 turnovers).[8]

The poor performance of Hb was somewhat unexpected in light of the

high reactivity of the structurally related Mb in this transformation

(1,650 TON)[8] and it could stem from unfavorable allosteric effects in

the case of the oligomeric protein. In contrast, wild-type CYP102A1

show considerably higher azide oxidation activity, supporting 1,400

catalytic turnovers for formation of the desired product 2a and

providing a product conversion ratio of 18% (Figure 1b-c) The

reaction also produced small amounts of benzylamine 2b (0.5%) and

N-benzyl-benzylimine (2c, <3%), as observed in the Mb-catalyzed

reaction.[8] Formation of the benzylamine 2b byproduct was ascribed

to an unproductive pathway resulting from decomposition of the azide

followed by reduction and protonation of the resulting nitrene-heme

intermediate, whereas 2c originates from condensation of 2b with the

aldehyde product.[8] Importantly, these initial results clearly evidenced

[a] Dr. Simone Giovani, Hanan Alwaseem, Prof. Dr. Rudi Fasan

Department of Chemistry, University of Rochester

120 Trustee Road, Rochester, NY 14627, United States

E-mail: rfasan@ur.rochester.edu

Supporting information for this article is given via a link at the end of

the document.

COMMUNICATION

the impact of the heme coordination environment on the reactivity of

the different hemoproteins toward organic azides, thereby expanding

previous findings from our group.[7e] Indeed, while HRP was

previously found to promote the intramolecular C−H amination of

arylsulfonyl azides with comparable or higher activity than P450s or

Mb,[5a, 5b, 7e] this enzyme shows significantly reduced activity toward

azide-to-aldehyde conversion compared to the latter hemoproteins.

Conversely, the data in Figure 1b-c suggested that, despite the

inherent differences between their active sites, the thiolate-bound

heme in the P450 is equally efficient as the histidinyl-bound heme

cofactor in Mb in promoting the oxidative deamination of alkyl azide 1

(1,400 TON vs. 1,650 TON for wild-type Mb[8]).

Figure 1. Catalytic activity of various hemoproteins in the oxidative deamination

of benzyl azide 1 to benzaldehyde 2a (a). (b-c) Reactions (400 L) were

conducted under anaerobic conditions with 10 mM BnN3, 10 mM Na2S2O4, and

1 M protein for 24 hours at room temperature. TON = nmol aldehyde / nmol

catalyst. Product yield is based on conversion of 1 to 2a as determined by gas

chromatography. (d) Reaction rates as determined over the first 10 minutes of

the reaction. (e) TON vs. pH plot for FL#62 reaction.

Given the promising activity of wild-type CYP102A1, we next

extended our investigations to two engineered variants of this enzyme,

namely CYP102A1(F87A) and FL#62. The former variant was chosen

because of the well documented ability of the F87A mutation in

expanding the substrate profile of CYP102A1 toward non-native

substrates in the context of monooxygenation reactions.[11, 15] In

addition to possessing a broad substrate profile,[16] the FL#62 variant,

which carries 6 active site mutations and 10 additional mutations

within its heme domain, was chosen because of its high reactivity in

the context of the C−H amination of azide-based reagents.[5a, 5b] As

shown by the data summarized in Figure 1, CYP102A1(F87A)

showed only slightly improved TON values for the conversion of

benzyl azide 1 into benzaldehyde 2a compared to the wild-type P450

(1,600 vs. 1,400 turnovers). In contrast, FL#62 was found to catalyze

this transformation with considerably higher efficiency (Figure 1b-c),

supporting 9,600 catalytic turnovers and providing nearly quantitative

product conversion (92%) at a catalyst loading of merely 0.001 mol%.

At lower catalyst loadings (0.0005 mol%), a TON value as high as

11,300 was measured for this enzyme. Notably, the FL#62 reaction

was found to proceed also with excellent chemoselectivity, leading to

the clean formation of 2a without producing detectable amounts of the

2b or 2c byproducts. Altogether, these properties make FL#62 not

only a considerably more efficient catalytic system for azide-to-

aldehyde conversion than synthetic Mo-based catalysts (50-99%

conversions using 10 mol% at 100°C in water/toluene mixture) [9a], but

Table 1. Substrate scope for FL#62-catalyzed oxidative deamination of alkyl

azides. Reaction conditions: 10 mM azide, 1 M FL#62, 10 mM Na2S2O4.

Prod. Structure R1 R2 % conv.

(% select.) TON

3b

H p-CH3 58 (94) 6,000

4b H p-

OCH3 99 (99) 9,440

5b H p-

NO2 25 (84) 2,670

6b H p-CF3 69 (93) 6,820

7b H o-CH3 22 (79) 2,000

8b H o-F 96 (98) 9,660

9b H o-

NO2 1 (18) 60

10b H m-

NO2 67 (94) 6,600

11b H o,p- di-F

51 (90) 5,850

12b H m,m-

di-CH3

36 (88) 3,540

13b H o,o- di-Cl

7 (72) 720

14b CH3 Ph 5 (51) 490

15b CF3 Ph 76 (98) 7,470

16b CO2Me Ph 49 (91) 4,650

17b

70 (94) 7,480

18b

97 (98) 9,500

19b

19 (85) 1,970

COMMUNICATION

also a superior biocatalyst compared to the best engineered Mb

variant (Mb(H64V,V68A)) identified in the course of our previous

studies.[8]

Further characterization studies revealed a noticeable effect of pH

on FL#62 azide oxidative deamination activity, with maximal TON

values being achieved around neutral pH (Figure 1e). A similar pH

dependence was observed for the Mb-catalyzed reaction.[8] From time

course experiments, the FL#62-catalyzed conversion of 1 to 2b was

determined to proceed with a rate of approximately 14 turnovers min-

1 over the first 10 minutes (Figure 1d). This rate is lower than that

observed for the Mb-based catalysts (250 min-1)[8], but the FL#62-

catalyzed reaction proceeds with higher selectivity (i.e., no 2b/2c

formation) in addition to the higher TON. The initial product formation

rates for FL#62 and the other hemoproteins investigated also

correlated well with the TON values observed in the corresponding

reactions (Figure 1b and 1d).

Next, the substrate scope of the FL#62 biocatalyst was

investigated using a wide panel of different organic azides. As shown

in Table 1, the majority of these compounds could be readily oxidized

to the corresponding aldehydes in good to excellent GC yields (49-

99%). In these reactions, the P450 was determined to support from

4,650 to 9,660 TON. In addition, the reaction proceeds with high

selectivity (>90-95%) in particular for the alkyl azides most efficiently

processed by the enzyme. The viable substrates include variously

mono- and disubstituted benzyl azide derivatives (3a, 4a, 6a, 8a, 10a,

11a), (hetero)aryl-substituted primary alkyl azides (17a, 18a), and

secondary alkyl azides (15a ,16a), which support the broad substrate

scope of the FL#62 catalyst. Modest (20-30%) to low (<20%) product

conversions were observed instead with benzyl azide derivatives that

carry one or two substituents at the ortho position(s) (i.e., 7b, 9b and

13b). These results suggest a negative effect on the enzymatic

transformation of increased steric hindrance in proximity to the azido

group. Whereas the conversion yields of these P450-catalyzed

reactions are consistently higher than those achieved with engineered

myoglobins,[8] particularly striking is the considerably higher efficiency

of the P450 catalyst in the synthesis of ketones 15b and 16b from the

secondary alkyl azides 15a and 16a, respectively. Indeed, while this

reaction proceeds inefficiently in the presence of the Mb catalyst (280-

400 TON, <2% conversion)[8], it is effectively catalyzed by the

engineered P450 (4,650-7,470 TON), resulting in much improved

product conversions (49-76%).

To further evaluate the synthetic utility of FL#62 in the context of

this reaction, a larger scale reaction with benzyl azide 1 (50 mg, 0.38

mmol) was carried out in the presence of 0.05 mol% FL#62 in

phosphate buffer (pH 7.0) at room temperature. After a simple

extraction and purification step, the desired benzaldehyde product 2a

was obtained in 87% isolated yield (35 mg, 0.33 mmol), thus

supporting the scalability of this biocatalytic transformation.

In terms of mechanism, we previously proposed that this reaction

involves heme-catalyzed decomposition of the heme-bound alkyl

azide to give an imido-iron(IV) intermediate. The latter then undergoes

a tautomerization to an imine-iron(II) complex, followed by hydrolysis

of the imine (in either free or heme-bound form) to yield the aldehyde

product (Figure 2). Experimental evidence in support of the imine

tautomerization step included the observation of a significant primary

kinetic isotope effect upon H→D substitution at the level of the

carbon in the azide substrate.[8] Interestingly, a related imine

intermediate was recently invoked in the azide-to-nitrile conversion

catalyzed by non-heme iron-dependent dioxygenases.[17]

Furthermore, 18O labelling experiments indicated that the oxygen

Figure 2. Proposed mechanism and catalytic steps for the P450-catalyzed

oxidation of alkyl azides to aldehydes.

atom in the aldehyde product derives from a hydrolytic process.[8] In

light of the higher catalytic activity (TON) and slower rate of the P450-

catalyzed reaction versus the Mb-catalyzed one, we thus wondered

whether these biocatalytic transformations shared an analogous

mechanism or not. To this end, we measured the KIE for the FL#62-

catalyzed oxidative deamination of benzyl azide (1) from competition

experiments in the presence of an equimolar concentration of the

deuterated analog, d2-1. LC-MS analysis of this reaction (SI Figure

S3a) yielded a KIE value (kH/kD) of 1.9 ± 0.2 at 22°C, which is

comparable to that observed in the presence of Mb(H64V,V68A) as

the catalyst (kH/kD = 1.7 ± 0.3)[8]. In addition, the FL#62-catalyzed

conversion of benzyl azide (1) in the presence of 18O-labeled water

resulted in the formation of 18O-labeled aldehyde product 2a(18O) (SI

Figure S3b), as observed for the Mb-catalyzed reaction. Altogether,

these results hint at important mechanistic similarities between the

two systems, although differences between them are also apparent.

Indeed, the negligible formation of the amine byproduct in the FL#62

reactions across all the tested azide substrates suggests that the

unproductive pathway leading to this reduction reduction is largely

disfavored in the case of the P450 system. In addition, the significantly

higher (10- to 15-fold) conversions and TON values measured for the

FL#62-catalyzed formation of 15b and 16b versus 14b (Table 1)

suggest that the P450 catalyst is significantly more sensitive to an

increase in the acidity of the alpha protons compared to the Mb

catalyst (3- to 4-fold higher TON for 15b and 16b, respectively,

compared to 14b).[8]

Kinetic experiments with wild-type CYP102A1,

CYP102A1(F87A), and FL#62 were carried out to gain further insights

into the catalytic properties of these enzymes. In the reaction with

model substrate 1, all of these P450s were found to follow Michaelis-

Menten kinetics (Figure 3). Similar kinetic parameters (KM, kcat) were

determined for the parent enzyme and its single mutant variant,

CYP102A1(F87A), which is in line with their similar performance in

terms of TON and product conversion (Table 1). Conversely, the

superior catalytic performance of FL#62 is reflected by an 8-fold

higher catalytic efficiency (kcat/KM = 4.6 x 102 M-1 s-1) compared to the

parent enzyme and F87A variant, which derives as a result of a two-

fold lower KM and a four-fold faster turnover number, kcat (Table 2).

While the catalytic efficiency of FL#62 in this non-native reaction is

significantly lower than that of CYP102A1 for the hydroxylation of fatty

COMMUNICATION

acids (kcat/KM ~ 105 M s-1)[18]], but it falls in the same range of those

exhibited by P450s involved in drug and hormone metabolism (e.g.,

P4501B1/retinol, kcat/KM ~ 7 x 103 M s-1)[19] For all the P450s,

incubation with benzyl azide resulted in a small (<5-10%) but

detectable heme spin shift (SI Figure S4), which is indicative of the

displacement of the axial water ligand on the heme by the bound

substrate. Titration experiments revealed a higher binding affinity of

FL#62 for benzyl azide as compared to wild type CYP102A1 and

CYP102A1(F87A) (KD of 68 M vs. 93 and 84 M, respectively; Table

2; SI Figure S4).

Another interesting result emerging from our substrate scope

investigations with FL#62 is the noticeable difference between the

TON values associated with the synthesis of electrondeficient benzyl

aldehydes (5b; 2,670 TON) as compared to electronrich ones (4b;

9,440). To elucidate the basis of this trend, we extended the substrate

binding experiments and Michaelis-Menten analyses to the FL#62-

catalyzed oxidative deamination of p-nitro-benzyl azide (5a) and p-

methoxy-benzyl azide (4a). While p-nitro-benzyl azide has weaker

affinity for FL#62 compared to benzyl azide and p-methoxy-benzyl

azide (Table 2), no significant differences were found among the

kinetic parameters (KM, kcat) and catalytic efficiency (kcat/KM) of the

enzyme for the transformation of these substrates, suggesting that

other factors must be at the basis of the less efficient conversion of

5a as compared to 4a and 1.

Figure 3. Overlay plot of initial velocity (V0) versus substrate concentration for

the reactions of CYP102A1 and its variants with selected alkyl azide substrates.

The corresponding kinetic parameters (kcat, KM, kcat/KM) are reported in Table 2.

Table 2. Substrate binding affinity and Michaelis-Menten parameters

corresponding to CYP102A1, CYP102A1(F87A), FL#62 and selected alkyl

azide substrates. Equilibrium dissociation constants (KD) were calculated from

substrate-induced heme spin shift experiments (SI Figure S4). The kinetic

parameters (kcat, KM, kcat/KM) were calculated via non-linear fitting of the V0 vs.

concentration curves (Figure 3) to the Michaelis-Menten equation.

Enzyme Substrate

KD (M)

KM (M)

kcat (min-1)

kcat/KM (M-1

*s-1)

CYP102A1 BnN3

(1) 93

(± 9) 1.8 x 10-2

(± 0.7) 66

(± 13) 61

(± 27)

CYP102A1 (F87A)

BnN3

(1) 84

(± 13) 1.9 x 10-2

(± 0.5)

75 (± 11)

66 (± 20)

FL#62 BnN3

(1) 68

(± 10) 9.2 x 10-3

(± 1.9)

255 (± 20)

4.6 x 102

(± 1.0)

FL#62 p-OMe-BnN3

(4a)

107 (± 19)

8.3 x 10-3

(± 1.0)

209 (± 9)

4.2 x 102

(± 0.5)

FL#62 p-NO2-BnN3 (5a)

259 (± 36)

1.1 x 10−2 (± 0.2)

329 (± 29)

5.0 x 102

(± 1.0)

To further examine this aspect, we performed product inhibition

experiments in which reaction mixtures containing FL#62 and 1, 4a,

or 5a as the substrates were spiked with increasing concentrations (0-

10 mM) of the corresponding aldehyde products. Interestingly, the

addition of para-nitro-benzaldehyde (5b) significantly reduced the

conversion of 5a (Figure 4a), evidencing the occurrence of product

inhibition. In contrast, no product inhibition was observed within the

concentration range tested for both benzyl azide (1) (SI Figure S5)

and para-methoxy-benzyl azide (4b) (Figure 4b), thus explaining the

greater yields observed in the presence of these substrates (Table 1).

In conclusion, our results demonstrate that cytochrome P450s are

promising catalysts for the synthesis of aromatic aldehydes via the

oxidative deamination of alkyl azides. In particular, we established

that the engineered CYP102A1 variant FL#62 represents a superior

biocatalyst for this transformation compared to other heme-containing

enzymes and the previously reported Mb catalysts. In addition to

excellent chemoselectivity and high catalytic activity (up to 11,300

TON) across a broad range of alkyl azides, the enhanced reactivity of

this P450 enabled extension of this transformation to the synthesis of

ketones from secondary azides. These studies contributed valuable

insights into the differential reactivity of hemoproteins, and engineered

variants thereof, in the context of non-native reactions, which is

expected to aid the future development of biocatalysts for a growing

number of synthetically useful transformations not found in nature.

Figure 4. Product inhibition experiments for (a) the reaction of FL#62 and p-

nitro-benzyl azide (5a) in the presence of added p-nitro-benzaldehyde (5b) and

(b) the reaction of FL#62 and p-methoxy-benzyl azide (4a) in the presence of

added p-methoxy-benzaldehyde (4b). See caption of SI Figure S5 for further

details.

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Acknowledgements

This work was supported by the U.S. National Institute of Health

grant GM098628. MS instrumentation was supported by the U.S.

NSF grant CHE-0946653.

Keywords: azide oxidative deamination • aldehyde synthesis •

P450 BM-3 • protein engineering • hemoprotein

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COMMUNICATION

Entry for the Table of Contents

COMMUNICATION

Simone Giovani, Rudi Fasan*

Page No. – Page No. Aldehyde and ketone synthesis via P450-catalyzed oxidative deamination of alkyl azides

New reaction for P450s: An engineered variant of CYP102A1 efficiently catalyzes the oxidative deamination of primary alkyl azides to yield aldehydes, exhibiting a broad substrate scope along with high catalytic activity and excellent chemoselectivity. The enhanced reactivity of this enzyme also enabled the conversion of selected secondary alkyl azides to ketones.

S1

Table of contents:

Supplementary Figure S1-S5 Pages S2-S6

Experimental Procedures Pages S7-S9

References Page S10

S2

Figure S1. Kinetic isotope effect (KIE) experiments. a) GC-MS spectrum of the mixture of benzyl

azide (1; m/z 133.2) and deuterated benzyl azide (d2-1; m/z 135.2) prior to the reaction with P450

FL#62 (1 : d2-1 ratio of 0.9 : 1). b) GC-MS spectrum of the benzaldehyde (2a) and deuterated

benzaldehyde (d-2a) products after the reaction of P450 FL#62 with the 1 / d2-1 mixture. The KIE

value was calculated from the integrated MS signals corresponding to the molecular ions of 2a

(m/z 106.1) and d-2a (m/z 107.1). Reaction conditions: 400 L-scale reaction containing 5 M

P450 FL#62, 10 mM 1 + d2-1, 10 mM Na2S2O4 in phosphate buffer (pH 7.0) at room temperature

for 6 hours.

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Figure S2. 18O labeling experiments. GC-MS spectrum of benzaldehyde (2a) and 18O-containing

benzaldehyde (2a(18O)) formed upon the reaction of P450 FL#62 with benzyl azide (1) in the

presence of 50% H218O under standard reaction conditions (1 M protein, 10 mM benzyl azide,

10 mM sodium dithionite in KPi (50% H218O) at pH 7.0 (not adjusted), room temperature, 24

hours). The isotopic distribution (62 % 16O, 38% 18O) was calculated from integration of the MS

signals corresponding to the molecular ions of 2a (m/z 106.1) and 2a(18O) (m/z 108.1).

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Figure S3. Isotopic labeling studies. a) Measurement of kinetic isotope effect (KIE) for H/D

substitution of protons in FL#62-catalyzed oxidation of benzyl azide. b) 18O labelling experiment

in FL#62 reaction with benzyl azide in the presence of H218O.

S5

Figure S4. Substrate binding studies with selected P450 enzymes and azide substrates. Left panel:

overlay of the visible absorbance spectrum before (gray line) and after (red line) addition of the

indicated substrate (0.5 mM), illustrating the substrate-induced shift of the heme spin state

equilibrium. Right panel: plot of substrate-induced heme spin shift versus substrate concentration.

The equilibrium dissociation constant (KD) for the enzyme−substrate complex was calculated via

nonlinear fitting of the experimental data (dots) to a noncooperative 1:1 binding model equation

(solid line).

A) CYP102A1 and benzyl azide (1)

B) CYP102A1(F87A) and benzyl azide (1)

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C) FL#62 and benzyl azide (1)

D) FL#62 and p-methoxy-benzyl azide (4a)

E) FL#62 and p-nitro-benzyl azide (5a)

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Figure S5. Product inhibition experiment for the FL#62-catalyzed conversion of benzyl azide (1)

to benzaldehyde (2a). The amount of benzaldehyde product (2a) formed in the presence of

exogenously added benzaldehyde (1, 2, 5, and 10 mM) is compared to that of a reference reaction

containing no exogenous benzaldehyde. The amounts of the aldehyde or azide species in the graph

are reported as relative to the % conversion and % residual substrate, respectively, in the reference

reaction. "% aldehyde (calc)" refers to the relative amount of total aldehyde product expected in

the absence of product inhibition (= aldehyde formed in the reference reaction + added aldehyde).

"% aldehyde (exp)" refers to the experimentally observed total aldehyde product in the reaction.

"% aldehyde (new)" refers to the relative amount of newly formed aldehyde product. "% azide

(res)" refers to the relative amount of residual azide substrate at the end of the reaction.

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Experimental Procedures:

Reagents and Analytical Methods. All the chemicals and reagents were purchased from

commercial suppliers (Sigma-Aldrich, ACS Scientific, Acros, VWR, Alfa Aesar) and used without

any further purification, unless otherwise stated. Bovine catalase, human hemoglobin, and

horseradish peroxidase were purchased from Sigma Aldrich. 18O-Water (Normalized, 97.4 atom

%) for isotopic labeling experiments was purchased from Icon Isotopes. All dry reactions were

carried out under argon atmosphere in oven-dried glassware with magnetic stirring using standard

gas-light syringes, cannulae and septa. Gas chromatography (GC) analyses were carried out using

a Shimadzu GC-2010 gas chromatograph equipped with a FID detector and an Agilent J&W GC

Chiral Cyclosil-B Column (30 m x 0.25 mm x 0.25 μm film). Separation method: 1 μL injection,

injector temp.: 200 ºC, detector temp: 300 ºC. Gradient: column temperature set at 60 ºC for 0.10

min, then to 100 ºC at 60 ºC/min, to 200 oC at 8 ºC/min, to 230 °C at 30 °C/min and then at 230

°C for 1.0 min. Total run time was 15.27 min. UV-Vis spectra were recorded on a Shimadzu UV-

2401PC UV-VIS spectrophotometer, Wavelength Range (nm) from 700 to 300, Scan Speed: Fast,

Sampling Interval (nm): 1.0, Scan Mode: Single.

Substrates and reaction products. The azide substrates and aldehyde/ketone products were

prepared as described in our previous report[1], which also provides full characterization data

(1H/13C(/19F) NMR, MS) for these compounds.

Protein expression and purification. Wild-type CYP102A1 and its engineered variants were

expressed from pCWori-based plasmids containing the P450 gene under the control of an IPTG-

inducible promoter (BamHI/EcoRI cassette) according to procedures described previously.[2]

FL#62[3] contains the following mutations: V78A, F81S, A82V, F87A, P142S, T175I, A180T,

A184V, A197V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V, where the active site

mutations are underlined. Typically, cultures of recombinant DH5α cells were grown at 37 °C (200

rpm) in Terrific Broth (TB) medium supplemented with ampicillin (100 mg/L) until OD600 reached

1.0 and then induced with 0.25 mM β-D-1-thiogalactopyranoside (IPTG) and 0.3 mM δ-

aminolevulinic acid (ALA). After induction, cultures were shaken at 150 rpm and 27 °C and

harvested after 20 h by centrifugation at 4000 rpm at 4 °C. Cell lysates were prepared by sonication

and loaded on Q Sepharose resin. P450s were eluted using 20 mM Tris, 340 mM NaCl, pH 8.0.

After buffer exchange (50 mM potassium phosphate buffer, pH 8.0), the enzymes were stored at

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−80 °C. P450 concentration was determined from CO-binding difference spectra (450–490 = 91 000

M–1 cm–1).

Enzymatic reactions. The enzymatic reactions were typically carried out at a 400 μL scale in KPi

buffer (50 mM) using 1 μM catalyst (standard concentration for all the enzymes) and 10 mM

sodium dithionite. A solution containing sodium dithionite (100 mM stock solution) in KPi buffer

(50 mM, pH 7.0) was degassed by bubbling argon into the mixture for 3 min in a sealed vial. A

separate vial containing the catalyst was carefully degassed in a similar manner. The solution was

transferred to the catalyst-containing vial via cannulation. Reaction was initiated by addition of a

10 mM solution of the appropriate azide (200 mM stock solution in methanol, final percentage of

methanol 5% v/v), with a syringe, and the reaction mixture was stirred for 24 h at 25 °C, under

positive argon pressure.

Product analysis. The reactions were analyzed by adding 20 µL of internal standard

(benzodioxole, 5 mM in methanol) to the reaction mixture, followed by extraction with 400 µL of

dichloromethane and analyzed by GC-FID as described previously.[1] TON = nmol aldehyde /

nmol catalyst. Product yield is based on conversion of the appropriate azide to the corresponding

aldehyde as determined by gas chromatography. Calibration curves for quantification of the

different products were constructed using authentic standards produced synthetically as described

previously.[1] All measurements were performed at least in duplicate.

18O labeling experiments. For the 18O incorporation experiments, reactions were carried out at a

400 µL scale in KPi buffer (pH 7.0) containing 50% H218O using 1 M P450 FL#62, 10 mM

sodium dithionite, and 10 mM benzyl azide in methanol (5% v/v) under an argon atmosphere. The

reactions were gently stirred 24 hours at 25 °C, then added of 20 L of internal standard

(benzodioxole, 5 mM in methanol). The products were extracted with 400 L of dichloromethane

and analyzed by GC-MS as described above.

Kinetic analyses. Reactions were carried out on a 400 L scale using the enzyme at a fixed

concentration of 1 M, 10 mM sodium dithionite, and substrate 1, 4a, or 5a at varying

concentration (1, 2, 5, 10, 15, 20, 30 mM) in KPi buffer (50 mM, pH 7.0). Initial velocity (V0) was

measured based on the amount of aldehyde product (2a, 4b, or 5b, respectively) formed after 90

minutes. The kinetic parameters Vmax, kcat , and KM were obtained by fitting the resulting plot of

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initial velocity (V0) vs. substrate concentrations ([S]) to the Michaelis-Menten equation using

software GraphPad Prism (version 6.01).

Heme spin shift experiments. Substrate binding experiments were performed using 3 μM purified

P450 in potassium phosphate buffer (50 mM, pH 8.0) by titrating increasing amounts of alkyl azide

(10 μM to 1 mM) from an ethanol stock solution (50 mM). At each concentration, a difference

spectrum from 350 to 500 nm was recorded and binding curves were generated by plotting the

change in absorbance at 390 and 420 nm corresponding to the high-spin and low-spin state of the

enzyme, respectively, against the substrate concentration. KD values were calculated using Graph

Pad Prism via nonlinear fitting of the experimental binding curves to an equation describing a

standard 1:1 binding interaction. Reported mean and standard deviation values were calculated

from experiments performed in duplicate.

Product inhibition experiments. Product inhibition experiments were carried out using 10 mM

substrate (1, 4a, or 5a), 1 M FL#62, and 10 mM Na2S2O4 in KPi buffer (50 mM, pH 7.0). Each

reaction mixture was added with increasing amounts (1, 2, 5, 10 mM) of the corresponding

aldehyde product (2a, 4b, and 5b, respectively). A reference reaction was carried out under

identical conditions but without the addition of the product. The reactions were gently stirred 24

hours at 25 °C, then added of 20 L of internal standard (benzodioxole, 5 mM in methanol). The

products were extracted with 400 L of dichloromethane and analyzed by GC-MS as described

above. The occurrence of product inhibition was determined by comparing the theoretical maximal

amount of aldehyde product (given by the amount of aldehyde produced in the reference reaction

plus the amount of exogenous aldehyde product added to the reaction) with the actual amount of

aldehyde produced in the reaction.

S11

References

[1] S. Giovani, R. Singh, R. Fasan, Chem. Sci. 2016, 7, 234-239.

[2] K. Zhang, S. El Damaty, R. Fasan, J. Am. Chem. Soc. 2011, 133, 3242-3245.

[3] K. D. Zhang, B. M. Shafer, M. D. Demars, H. A. Stern, R. Fasan, J. Am. Chem. Soc. 2012, 134,

18695-18704.